The maintenance of bone mass is essential for the prevention of osteoporosis and fractures in the elderly. Besides anti-resorptive therapies, the only approved anabolic therapy is intermittent PTH, the mechanism of action of which is still incompletely understood. We have identified Zfp521, a 30 zinc-finger containing protein, as a prominent player in the transcriptional processes that contribute to bone formation (2). Zfp521 expression in cultured osteoblasts (OBs) increases with time, and is regulated by BMP-2 and PTH/PTHrP. In vivo, targeted over-expression of Zfp521 in mature OBs in transgenic mice resulted in an increase in bone formation (2). In contrast, osteoblast-targeted Zfp521-/- mice are osteopenic with impaired maturation of OBs, and decreased bone formation. Furthermore Zfp521 binds Runx2 and Ebf1 and repress their transcriptional activity as well as that of CREB on the Rankl promoter, repressing OB-induced bone resorption. Thus, our preliminary data shows that Zfp521 1) interacts with several key OB transcription factors, 2) responds to several key regulators of bone formation and 3) affects bone formation and bone mass when over-expressed or deleted in vivo in mice. This establishes Zfp521 as a novel and important contributor to the regulation of bone formation and homeostasis, and one that deserves further exploration. Accordingly, we propose to further explore the mechanism by which Zfp521 affects OB differentiation and bone homeostasis, focusing on its interplay with Runx2, Ebf1 and CREB. Since we have generated significant preliminary data and have already generated most of the genetic models required for this investigation (OG2-Zfp521; 2.3kb-Col1a1-Zfp521; both global Zfp521-/- and floxed Zfp521, as well as the OC- Cre-Zfp521 and Osx-Cre-Zfp521 lines, Ebf1 -/- and floxed Ebf1, 2.3kb-Col1a1-Ebf1), the specific aims of this research proposal are: Aim1: Further analyze the effects of full and OB-targeted conditional, stage-specific deletion of Zfp521 on OB differentiation and bone formation in vivo and in vitro; Aim2: Further characterize the molecular mechanisms by which Zfp521 exerts its effects on transcription factors in OBs, affecting bone formation, with particular emphasis on its interplay with Runx2 , Ebf1 and CREB on target genes. Aim3: Identify Zfp521 new partners, binding sites and target genes by genome-wide screening Functional characterization of this newly discovered co-regulator of bone formation will advance our understanding of key physiological processes that regulate bone homeostasis, ultimately allowing the exploration of novel pathways for drug discovery in osteoporosis and other low bone mass syndromes.